In order to further our understanding of the functional relationships between flavin and metal in "step-down" electron transport systems, we propose to continue our studies of the soluble FAD-FMN-Fe/S- heme enzyme, NADPH-sulfite reductase of enterobacteria. Using the holoenzyme, and an iron-free flavoprotein moiety and flavin-free hemoprotein moiety derived from it by either urea dissociation or mutation, we shall study the mechanism of flavin redox cycling in the presence and absence of metal components. We shall also utilize enzyme from which the FMN, but not the FAD, has been specifically removed, in order to ascertain the importance of flavin-flavin interaction in the electron transfer process. Our principal tools will be rapid reaction kinetic experiments, utilizing both absorption spectroscopy (stopped- flow) and EPR (rapid freeze). Titration experiments will be performed, along with measurement of the potentials of the various enzyme carriers. We shall utilize available complexes between enzyme and sulfite or reduction products thereof to study the nature and role of possible intermediates in the six-electron transfer reaction (reduction of SO3 equals to S equals) catalyzed by the enzyme. We shall also investigate three "dissimilatory" sulfite reductases which appear to "leak" compounds of intermediate oxidation state during the course of sulfite reduction to sulfide. We shall continue our studies of the structure and biological role of the novel heme prosthetic group of sulfite reductase, "siroheme", and initiate investigations into its possible model of biosynthesis and relationship to other tetrapyrrole prosthetic groups. Having recently found that siroheme is the prosthetic group of a second six-electron reductase enzyme, spinach nitrite reductase, we shall isolate and study nitrite reductases from several sources in order to establish the generality of this finding.